Non-invasive sensing of free metal ions using ion chemical exchange saturation transfer

ABSTRACT

The invention features a novel non-invasive approach for imaging, detecting and/or sensing metal ions with improved sensitivity and specificity in a biological sample or tissue. In certain embodiments, the invention provides a MR contrast-based approach for imaging, detecting and/or sensing metal ions in the biological sample/tissue containing various background ions by using 19F-based chemical exchange saturation transfer (CEST) technique.

RELATED APPLICATIONS

This application claims the benefit of priority of U.S. ProvisionalApplication No. 61/813,881, filed Apr. 19, 2013, which is herebyexpressly incorporated by reference in its entirety.

STATEMENT REGARDING FEDERALLY-SPONSORED RESEARCH

The present invention was supported by grants from the NationalInstitute of Health (NIH), grant numbers EB 012590 and EB 007825. TheU.S. Government may have certain rights to the present invention.

FIELD OF INVENTION

The present invention generally relates to methods of magnetic resonanceimaging (MRI), more particularly MRI that is based on chemical exchangesaturation transfer (CEST) and more specifically CEST based MRI thatutilizes ¹⁹F-based responsive MRI contrast agents.

BACKGROUND OF THE INVENTION

Metal ions play a crucial role in a myriad of biological processes, andthe ability to monitor real time changes in metal ion concentrations isessential for understanding a variety of physiological events.Alterations in cellular homeostasis of metal ions are connected to humandisorders and diseases including cancer, diabetes, and neurodegenerativedisease (Nat. Chem. Biol., 2008; 4: 168-175). As demonstrated in thecartoon of FIG. 1, metal ion signaling and homeostasis play an importantrole in many cellular processes. The main metal ions involved are themonovalent sodium and potassium, and the bivalent calcium, zincmagnesium and iron. Cells maintain healthy levels of the essential metalions using several classes of proteins. Currently, imaging dynamicchanges in metal ions levels is restricted to fluorescence-basedmethodologies which are limited by low tissue penetration and thus donot allow in vivo imaging of metal ions in deep tissues or organs.

Chemical exchange saturation transfer (CEST) MR imaging is a techniquein which low-concentration marker molecules are labeled by saturatingtheir exchangeable protons (e.g., hydroxyl, amine, amide, or iminoprotons) by radio-frequency (RF) irradiation. If such saturation can beachieved rapidly (i.e., before the proton exchanges), exchange of suchlabeled protons with water leads to progressive water saturation,allowing indirect detection of the solute via the water resonancethrough a decrease in signal intensity in MRI [Ward, K. M., Aletras, A.H. & Balaban, R. S. A new class of contrast agents for MRI based onproton chemical exchange dependent saturation transfer (CEST). J MagnReson 143, 79-87 (2000)].

Each CEST contrast agent can have a different saturation frequency,which depends on the chemical shift of the exchangeable proton. Themagnitude of proton transfer enhancement (PTE) due to this effect, andthe resulting signal reduction from equilibrium (S₀) to saturated (S),are given by [Goffeney, N., Butte, J. W., Duyn, J., Bryant, L. H., Jr. &van Zijl, P. C. Sensitive NMR detection of cationic-polymer-based genedelivery systems using saturation transfer via proton exchange. J AmChem Soc 123, 8628-8629 (2001)]:

$\begin{matrix}{{{PTE} = {\frac{{NM}_{w}\alpha \; k_{ex}}{{\left( {1 - x_{CA}} \right)R_{1\; {wat}}} + {x_{CA}k_{ex}}} \cdot \left\{ {1 - e^{{- {\lbrack{{{({1 - x_{CA}})}R_{1\; {wat}}} + {x_{CA}k_{ex}}}\rbrack}}t_{sat}}} \right\}}},} & \left\lbrack {{Eq}.\mspace{14mu} 1} \right\rbrack \\{{{and}\mspace{14mu} \left( {1 - {S_{sat}/S_{0}}} \right)} = {\frac{{PTE} \cdot \lbrack{CA}\rbrack}{2 \cdot \left\lbrack {H_{2}O} \right\rbrack}.}} & \left\lbrack {{Eq}.\mspace{14mu} 2} \right\rbrack\end{matrix}$

“CA” is the contrast agent containing multiple exchangeable protons,x_(CA) its fractional exchangeable proton concentration, α thesaturation efficiency, k the pseudo first-order rate constant, N thenumber of exchangeable protons per molecular weight unit, and M_(w) themolecular weight of the CA. The exponential term describes the effect ofback exchange and water longitudinal relaxation (R_(1wat)=1/T_(1wat)) onthe transfer during the RF saturation period (t_(sat)). This effect willbe bigger when protons exchange faster, but the catch is that saturationoccurs faster as well, which increases the radio-frequency power needed.In addition, the resonance of the particular protons must be wellseparated from water in the proton NMR spectrum, which requires a slowexchange on the NMR time scale. This condition means that the frequencydifference of the exchangeable protons with water is much larger thanthe exchange rate (Δω>k).

Thus, the CEST technology becomes more applicable at higher magneticfields or when using paramagnetic shift agents [Zhang, S., Merritt, M.,Woessner, D. E., Lenkinski, R. E. & Sherry, A. D. PARACEST agents:modulating MRI contrast via water proton exchange. Acc Chem Res 36,783-790 (2003)]. Any molecule that exhibits a significant PTE effect canbe classified as a CEST (contrast) agent. The concept of these agents asMR contrast agents is somewhat similar to the chemical amplification ofcolorimetric labels in in situ gene expression assays. CEST agents canbe detected by monitoring the water intensity as a function of thesaturation frequency, leading to a so-called z-spectrum. In suchspectra, the saturation effect of the contrast agent on the waterresonance is displayed as a function of irradiation frequency.

Since the first report of CEST contrast in 2000, CEST MR imaging hasbecome widely used MRI contrast mechanism (demonstrated in FIG. 2). FIG.2 shows that a CEST contrast is generated by the dynamic exchangeprocess between an exchangeable proton of a biomarker of interest andthe surrounding water protons. To detect the biomarkers, themagnetization of some of their exchangeable protons is nullified byapplying a selective radiofrequency saturation pulse at the specificresonance frequency (chemical shift) of the target protons. Due toexchange of the “saturated” agent protons with surrounding waterprotons, the net water signal is reduced thus enhancing the MRIcontrast.

CEST-MRI has been employed for many applications in molecular andcellular MRI (see, e.g., Bar-Shir, A. et al., J. Am Chem. Soc. 2013;Ratnakar, S. J. et al., J. Am Chem. Soc. 2012, 134, 5798; Liu, G. etal., Magn Reson Med. 2012, 67, 1106; Longo, D. L. et al., Magn ResonMed. 2012, doi: 10.1002/mrm. 24513; Li., Y. et al. Contrast Media MolImaging 2011, 6, 219; Aime, S. et al. Angew Chem Int Ed Engl 2005, 44,1813; Chan, K. W. et al., Nat Mat 2013, 12, 268; Liu, G. et al., NMR inBiomedicine 2013, doi: 10.1002/nbm.2899).

However, despite that recent advances in the field of molecular magneticresonance imaging (MRI) has led to the development of new strategies inthe design and synthesis of responsive MRI contrast agents for detectingbiologically relevant metal ions, the specificity and sensitivity ofthose probes is limited.

Therefore, there is a need for the development of novel methodology andresponsive MRI contrast agents for detecting biologically relevant metalions with improved specificity and sensitivity.

SUMMARY OF THE INVENTION

The present invention features a novel method of detecting biologicallyrelevant metal ions. In particular embodiments, the invention utilizeschemical exchange saturation transfer (CEST) MR technique for imaging ametal ion in a biological sample or tissue.

In one aspect, the invention provides a method of obtaining a magneticresonance (MR) image of a metal ion in a biological sample or tissue.The method comprises

a) introducing a ¹⁹F-based responsive magnetic resonance imaging (MRI)contrast agent to a biological sample or tissue containing the metalion; and

b) imaging the biological sample or tissue using a chemical exchangesaturation transfer (CEST)-based MRI technique.

In another aspect, the invention relates to a method of detecting orsensing a metal ion in a biological sample or tissue comprisingbackground ions. Specifically, the method comprises steps of

a) introducing to the biological sample or tissue ¹⁹F-based responsivemagnetic resonance imaging (MRI) contrast agents, wherein at least oneof the ¹⁹F-based responsive MRI contrast agents is bound to the metalion to produce a chelation complex;

b) radiofrequency (RF) labeling of ¹⁹F frequency in said chelationcomplex; and

c) detecting label transfer to ¹⁹F frequency in a ¹⁹F-based responsiveMRI contrast agent free of a metal ion chelation.

In certain embodiments, the method further includes a step of detectinga chemical shift change of ¹⁹F by using a ¹⁹F nuclear magnetic resonance(NMR) technique.

Specifically, provided herein is a method of detecting or sensing Ca²⁺in a biological sample or tissue comprising background ions. The methodcomprises steps of

a) introducing to the biological sample or tissue a ¹⁹F-derivative of1,2,-bis(o-aminophenoxy)ethane-N,N,—N′,N′, tetra-acetic acid (BAPTA), ora salt or ester thereof to obtain a chelation complex containing themetal ion and the ¹⁹F-derivative; and

b) detecting a chemical shift change of ¹⁹F through a ¹⁹F NMR.

As used herein, the background ions refer to other metal ions or othertypes of ions that are present in the biological sample or tissue, whichare not the interest for imaging/or detecting.

Also featured herein are kits for MRI imaging of free metal ions in abiological sample or tissue. The kit of the invention includes one ormore ¹⁹F-based responsive MRI contrast agents of the invention (detailsprovided infra.), and instructions for imaging free metal ions in thebiological sample or tissue.

Also featured are MRI methods that embody the use of the responsive MRIcontrast agents of the invention.

Other aspects and embodiments of the invention are discussed below.

BRIEF DESCRIPTION OF THE DRAWING

For a fuller understanding of the nature and desired objects of thepresent invention, reference is made to the following detaileddescription taken in conjunction with the accompanying drawing figureswherein like reference character denote corresponding parts throughoutthe several views and wherein:

FIG. 1 is a cartoon illustrating metal ion signaling and homeostasis inmany cellular processes.

FIG. 2 demonstrates the contrast mechanism of CEST-MRI.

FIG. 3 is a cartoon demonstrating a process involving transplantinghuman islet for diabetes therapy; the vast majority of the islets arebeta cells secreting insulin and the main ions to be detected todetermine their functionality are calcium and zinc.

FIGS. 4 a-b) show that metal ions (M²⁺) binding 5F-BAPTA: a) schematicdepiction of a dynamic exchange process between free 5F-BAPTA and bound[M²⁺-5F-BAPTA]; b) ¹⁹F NMR spectra (470 MHz) of 5F-BAPTA in the presenceof Mg²⁺, Zn²⁺, and Ca²⁺.

FIGS. 5 a-f) are ¹⁹F-iCEST Z-spectra of solutions containing 10 mM of5F-BAPTA and 50 μM of M²⁺ (Ca²⁺; Zn²⁺; and Mg²⁺) in 40 mM Hepes bufferwith the pH of the solutions adjusted to 7.2 (a-c) or 6.4 (d-f); dotsrepresent the raw experimental data; for Ca²⁺, lines represent Blochsimulations (two pool model) and arrows point to the frequency of the[Ca²⁺-5F-BAPTA] complex.

FIG. 6 shows MR images of Ca²⁺ with iCEST: ¹H-MRI, ¹⁹F-MRI, and iCEST(Δω=6.2 or 5.0 ppm) of M²⁺ solutions with pH values of 7.2 or 6.4; eachtube contains 10 mM of 5F-BAPTA and 50 μM of M²⁺.

FIGS. 7 a-b) show Ca²⁺ sensing using iCEST: a) χCa vs. MTR plot. b)Detection of 500 nM Ca²⁺ in the presence of 0.5 mM of 5F-BAPTA; inset inb) depicts ¹⁹F-MRI of the sample with an overlaid iCEST image; lines ina and b represent Bloch simulations.

FIGS. 8 a-d) are ¹⁹F-CEST spectra that show pH dependency of 5F-BAPTA:CEST-spectra of solutions containing 10 mM of 5F-BAPTA and 50 μM of Ca²⁺in 40 mM Hepes buffer at solutions with the pH adjusted to a) 5.6; b)6.0; c) 6.8; and d) 7.0; solid lines represent Bloch simulations (twopool model) and arrows point to the Δω of the [Ca²⁺-5F-BAPTA] complex.

FIG. 9 presents ¹⁹F-NMR spectra showing pH dependency of 5F-BAPTA: thespectra are for solutions containing Ca²⁺ (0.5 mM), 5F-BAPTA (5 mM), and5-Fluoro-Cytosine (5FC, 0.5 mM) as internal reference (set to −47 ppm)with 10% D₂O; the spectra were acquired at 470 MHz, with the peak of5-FC calibrated at −47 ppm.

FIG. 10 a-f): a-d) are ¹⁹F-iCEST Z-spectra of solutions containing a)Ca²⁺ (50 μM) and Mg²⁺ (200 μM); b) Mg²⁺ (200 μM); c) Ca²⁺ (50 μM); andd) Zn²⁺ (50 μM) at pH=7.2, 37° C., and 16.4 T; e-f) are MTR asymmetry(MTR_(asym)) plots of the background solutions are shown for 200 μM Mg²⁺(a & b) and for 50 μM Zn²⁺ (c & d); solid lines represent Blochsimulations.

FIG. 11 presents a graph showing pH dependency of iCEST. The dependencyof Δω between Ca²⁺-bound and free 5F-BAPTA as obtained from iCESTZ-spectra: the phantom includes 8 mm NMR tubes containing 10 mM of5F-BAPTA and 50 μM of M²⁺ in 40 mM Hepes buffer (pH=5.6-7.6); ¹⁹F-iCESTdata were acquired with B₁=3.6 μT/2000 ms at 37° C.; the Δω dependencybetween Ca²⁺-bound and free 5F-BAPTA was obtained from ¹⁹F-iCESTZ-spectra.

FIG. 12 a-g) show sensing Ca²⁺ using iCEST: a) alignment of four tubescontaining 5 mM of 5F-BAPTA (pH=7.2) and different molar fractions(χCa=1:250, 1:500, 1:1000, 1:0) between Ca²⁺ and 5F-BAPTA for thephantom (pH=7.2, 16.4 T, 37° C.); b) 1H-MR image, c) 19F-MR image; andd) overlay of ¹⁹F-iCEST image (Δω=6.2 ppm) on ¹⁹F-image; e) iCESTZ-spectra for a tube with χCa=1:1000; and f) iCEST Z-spectra for a tubewith χCa=1:500

Samples; solid lines represent Bloch simulations; g) plot of χCa vs.MTRasym for iCEST data acquired at B₁=3.6 μT, t_(sat)=1.5 s; a 5%threshold is shown as a gray dashed line, which is reached atχCa=1:2000.

FIGS. 13 a-c) demonstrate Ca²⁺ specificity of 5F-BAPTA in a Zn²⁺background.

FIGS. 14 a-c) demonstrate Ca²⁺ specificity of 5F-BAPTA in a Mg²⁺background.

FIG. 15 demonstrates the mechanism of CEST-MRI by using exchangeable¹⁹F-based molecule.

FIGS. 16 a-c): a) Schematic illustration of the chemical shift offsets (. . . ) at the 19F NMR spectrum of 5F-BAPTA upon chelation withdifferent bivalent cations; b) demonstration of the dynamic exchangeprocess between free and Ca²⁺-bound 5F-BAPTA; c) ¹⁹F-CEST spectra (solidlines) and MTRasym plots (dashed lines) for the complex [Ca²⁺-5F-BAPTA]at room temperature (RT) and 37° C. obtained at 11.7 T.

FIGS. 17 a-c): a) ¹⁹F MRI of tubes containing 10 mM of 5F-BAPTA obtainedat 16.4 T, tubes contained either no M²⁺ content or one of the ionsMg²⁺, Zn²⁺ or Ca²⁺ at 100 μM concentration; b) MTRasym maps (Δω=5.8 ppm)obtained from the 19F-CEST experiment of the tubes shown in a; c)MTRasym plots (dashed lines) of the Ca²⁺ containing tube.

FIGS. 18 a-c): a) Chemical structure of 5,5′,6,6′-tetrafluoro-BAPTA(TF-BAPTA). b) ¹⁹F NMR spectrum (470 MHz) of 5 mM TF-BAPTA in thepresence of 0.5 mM Zn²⁺ or Fe²⁺. c) ¹H MRI, ¹⁹F MRI, and iCEST (Aω=−2.8ppm or Aω=−18 ppm) overlaid on ¹⁹F MRI. The far right shows the ¹⁹Fmulticolor multi-ion iCEST image for 10 mM TF-BAPTA and 200 μM metal ion(i.e., Ca²⁺, Mg²⁺, Zn²⁺, Fe²⁺, Cu²⁺, Na⁺ or K⁺, as noted in FIG. 18cwith B1=3.6 T/2 s.

FIGS. 19 a-c): a-b) Corresponding ¹⁹F-iCEST spectra for the samplesshown in FIG. 18c , containing 10 mM of TF-BAPTA and 200 μM of Zn²⁺ (a,red) and Fe²⁺ (b, green). Circles represent the average experimentalsignal, solid lines represent Bloch simulations (two pool model). Arrowspoint to the Aω of the [M²⁺-5F-BAPTA] complex where M preferablyrepresents Zn²⁺ or Fe²⁺. c) ¹⁹F-iCEST spectra for a sample containingboth Zn²⁺ and Fe²⁺ (not shown in FIG. 18), with Bloch simulations (solidline) performed using a three-pool model.

DETAILED DESCRIPTION OF THE INVENTION

Fluorescent metal ion sensors have been developed for the purpose, forexample, studying intracellular calcium (R. Y Tsien, Biochemistry19(1980); 2396), which enables a better understanding of calcium signals(Cell Calcium 40 (2006) 561). Many metal ion sensors derive from thefoundational calcium sensor molecules, which were designed by tagging afluorophore onto the backbone of a metal chelator to elicit ametal-dependent fluorescence response (L. M. Hyman et al., CoordinationChemistry Reviews, 256 (2012), 2333-2356).

Among those calcium sensors, many share a chelating backbone of1,2-bis(o-aminophenoxy)ethane-N,N,—N′,N′-tetraacetic acid (BAPTA), withthe following structure:

(Biochemistry 19(1980); 2396). Currently, imaging dynamic changes inCa²⁺ levels is restricted to fluorescence based methodologies which arelimited by low tissue penetration and as a result do not allow in vivoCa²⁺ imaging in deep tissues (Mank, M. et al., Chem Rev 2008, 108, 1550;and Tsien, R. Y. Annu Rev Neurosci 1989, 12, 227). As of today, anon-invasive means of detecting free Ca²⁺ in a deep tissue remains aformidable challenge.

The present inventors discovered a novel approach for sensing thepresence of biologically relevant metal ions (specifically, Ca²⁺), byusing a strategy, in which the amplification effects of chemicalexchange saturation transfer (CEST) is combined with the broad range inchemical shifts found in ¹⁹F NMR to obtain MR images of the metal ions.The inventors exploited the chemical shift change (Δω) of ¹⁹F uponbinding of Ca²⁺ to a difluoro derivative of[1,2,-bis(oaminophenoxy)ethane-N,N,—N′,N′,tetra-acetic acid], e.g.,5F-BAPTA with the following structure:

2,2′,2″,2′″-(2,2′-(Ethane-1,2-diylbis(oxy))bis(4-fluoro-2,1-phenylene))bis(azanetriyl)tetraaceticacid (alias, 1,2-bis-[2-bis(carboxymethyl)amino-5-fluorophenoxy]ethane;“5F-BAPTA”).

FIG. 15 demonstrates the mechanism of CEST-MRI by using exchangeable¹⁹F-based molecule.

Specifically, the inventors applied a CEST MRI contrast mechanism to¹⁹F-modified M+-chelates. In a typical CEST MRI contrast mechanism, adynamic exchange process between radiofrequency labeled protons and bulkwater is exploited for contrast enhancement. In particular, theinventors employed a saturation transfer approach that couples ¹⁹F- andCEST-MRI for specifically sensing the presence of M²⁺ ions through theirsubstrate binding kinetics, an approach called ion CEST (iCEST). Usingradiofrequency (RF) labeling at the bound ion-¹⁹F frequency,(ω[M²⁺-chelate]), and detection of label transfer to the free chelate¹⁹F frequency, (ω-chelate) (0 ppm), the adopted approach is able toamplify the signal of bound the metal ions by a factor of hundreds,depend on T1 and the exchange rate.

The following chart illustrates the approach undertaken by the presentinventors:

The ¹⁹F-MRI approach offers advantages due to the high gyromagneticratio of ¹⁹F (94% of that of ¹H), 100% natural isotopic abundance of¹⁹F, and the negligible amount of naturally occurring fluorine in softbiological tissues, which results in “hot spot” images withoutbackground signal. In addition, the large range of ¹⁹F chemical shifts(about 20 times than that of ¹H) and the sensitivity of ¹⁹F chemicalshifts to the details of the local environment is another benefit foriCEST-based applications.

Moreover, by using the iCEST approach, the contrast from lowconcentration solutes [M+-chelate] is amplified through back-and-forwardchemical exchange and observed on the signal of the high concentrationfree chelate. Compared to the ¹H CEST imaging, which is based on water,the ¹⁹F based approach enables detection of metal ions at lowerconcentrations through simply reducing the free chelate concentrations,as the contrast is dependent on the ratio of ion to agent.

Additionally, the present disclosure also provides compositions andmethods that allow simultaneously detection of more than one metal ion(e.g., K⁺, Na⁺, Ca²⁺, Mg²⁺, Cu²⁺, Fe²⁺ and Zn²⁺). For example, by adding¹⁹F atoms to the 6 position of 5F-BAPTA to obtain TF-BAPTA (see, e.g.,FIG. 18a ), it became possible to detect both Zn²⁺ and Fe²⁺. Adding one¹⁹F atom to the BAPTA backbone dramatically changes the bindingproperties of TF-BAPTA (London et al., 1994, Am J Physiol 266:1313). Atthe same time, the added ¹⁹F-atom induces k_(ex) values that allowdetection of Zn²⁺ and Fe²⁺ with ¹⁹F-iCEST MRI. Advantageously, thespecificity of iCEST to simultaneously detect different metal ions usingthe same sensor provides a new ability to rationally design novel MRIprobes.

DEFINITIONS

Before further description of the invention, and in order that theinvention may be more readily understood, certain terms are firstdefined and collected here for convenience.

As used in the specification and claims, the singular form “a”, “an” and“the” include plural references unless the context clearly dictatesotherwise. For example, the term “a cell” includes a plurality of cells,including mixtures thereof. The term “a nucleic acid molecule” includesa plurality of nucleic acid molecules.

In this disclosure, “comprises,” “comprising,” “containing” and “having”and the like can have the meaning ascribed to them in U.S. patent lawand can mean “includes,” “including,” and the like; “consistingessentially of” or “consists essentially” likewise has the meaningascribed in U.S. patent law and the term is open-ended, allowing for thepresence of more than that which is recited so long as basic or novelcharacteristics of that which is recited is not changed by the presenceof more than that which is recited, but excludes prior art embodiments.

The term “administration” or “administering” includes routes ofintroducing the compound of the invention to a subject to perform theirintended function. Examples of routes of administration that may be usedinclude injection (subcutaneous, intravenous, parenterally,intraperitoneally, intrathecal), oral, inhalation, rectal andtransdermal. The pharmaceutical preparations may be given by formssuitable for each administration route. For example, these preparationsare administered in tablets or capsule form, by injection, inhalation,eye lotion, ointment, suppository, etc. administration by injection,infusion or inhalation; topical by lotion or ointment; and rectal bysuppositories. Oral administration is preferred. The injection can bebolus or can be continuous infusion. Depending on the route ofadministration, the compound of the invention can be coated with ordisposed in a selected material to protect it from natural conditionswhich may detrimentally affect its ability to perform its intendedfunction. The compound of the invention can be administered alone, or inconjunction with either another agent as described above or with apharmaceutically-acceptable carrier, or both. The compound of theinvention can be administered prior to the administration of the otheragent, simultaneously with the agent, or after the administration of theagent. Furthermore, the compound of the invention can also beadministered in a pro-drug form which is converted into its activemetabolite, or more active metabolite in vivo.

The phrase “in combination with” is intended to refer to all forms ofadministration that provide an a compound of the invention (e.g. acompound selected from any of the formulae described herein) togetherwith a second agent, such as a second compound selected from any of theformulae described herein, or an existing therapeutic agent used for aparticular disease or disorder, where the two are administeredconcurrently or sequentially in any order.

The term “alkyl” refers to the radical of saturated aliphatic groups,including straight-chain alkyl groups, branched-chain alkyl groups,cycloalkyl(alicyclic) groups, alkyl substituted cycloalkyl groups, andcycloalkyl substituted alkyl groups. Preferred alkyl groups have from 1to 8 carbon atoms in the alkyl backbone (e.g., C₁-C₈ alkyl). As will beclear from context, the term “alkyl” as used herein further includesdivalent alkylene groups (e.g., —(CH₂)_(n)—, wherein n is a positiveinteger, e.g., 1 to 8), and can further include oxygen, nitrogen, sulfuror phosphorous atoms replacing one or more carbons of the hydrocarbonbackbone, e.g., oxygen, nitrogen, sulfur or phosphorous atoms. Thus, forexample, in a compound of the formula:

if each R¹ is independently “C₁₋₃ alkyl”, then each R¹ is independentlyC₁₋₃ alkylene. For convenience, C₀alkyl used herein refers to a bond ora H atom.

Moreover, the term alkyl (or alkylene) as used throughout thespecification and sentences is intended to include both “unsubstitutedalkyls” and “substituted alkyls,” the latter of which refers to alkylmoieties having substituents replacing a hydrogen on one or more carbonsof the hydrocarbon backbone. Such substituents can include, for example,halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl,aminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato,phosphinato, cyano, amino (including alkyl amino, dialkylamino,arylamino, diarylamino, and alkylarylamino), acylamino (includingalkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino,imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates,sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety. Itwill be understood by those skilled in the art that the moietiessubstituted on the hydrocarbon chain can themselves be substituted, ifappropriate. Cycloalkyls can be further substituted, e.g., with thesubstituents described above. An “alkylaryl” moiety is an alkylsubstituted with an aryl (e.g., phenylmethyl(benzyl)). The term “alkyl”also includes unsaturated aliphatic groups analogous in length andpossible substitution to the alkyls described above, but that contain atleast one double or triple bond respectively.

The term “alkoxy” refer to a —O-alkyl group.

The term “aryl” as used herein, refers to the radical of aryl groups,including 5- and 6-membered single-ring aromatic groups that may includefrom zero to four heteroatoms, for example, benzene, pyrrole, furan,thiophene, imidazole, benzoxazole, benzothiazole, triazole, tetrazole,pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, and the like.Aryl groups also include polycyclic fused aromatic groups such asnaphthyl, quinolyl, indolyl, and the like. Those aryl groups havingheteroatoms in the ring structure may also be referred to as “arylheterocycles,” “heteroaryls” or “heteroaromatics.” The aromatic ring canbe substituted at one or more ring positions with such substituents asdescribed above, as for example, halogen, hydroxyl, alkoxy,alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl,aminocarbonyl, alkylthiocarbonyl, phosphate, phosphonato, phosphinato,cyano, amino (including alkyl amino, dialkylamino, arylamino,diarylamino, and alkylarylamino), acylamino (includingalkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino,imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates,sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety. Arylgroups can also be fused or bridged with alicyclic or heterocyclic ringswhich are not aromatic so as to form a polycycle (e.g., tetralin).

The term “heteroaryl” refers to an aromatic 5-8 membered monocyclic,8-12 membered bicyclic, or 11-14 membered tricyclic ring system having1-4 ring heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9heteroatoms if tricyclic, with said heteroatoms selected from O, N, andS, and the remainder ring atoms being carbon. Heteroaryl groups may beoptionally substituted with one or more substituents. Examples ofheteroaryl groups include, but are not limited to, pyridyl, furanyl,benzodioxolyl, thienyl, pyrrolyl, oxazolyl, oxadiazolyl, imidazolylthiazolyl, isoxazolyl, quinolinyl, pyrazolyl, isothiazolyl, pyridazinyl,pyrimidinyl, pyrazinyl, triazinyl, triazolyl, thiadiazolyl,isoquinolinyl, indazolyl, benzoxazolyl, benzofuryl, indolizinyl,imidazopyridyl, tetrazolyl, benzimidazolyl, benzothiazolyl,benzothiadiazolyl, benzoxadiazolyl, and indolyl. In one embodiment ofthe invention, heteroaryl refers to thienyl, furyl, pyridyl, or indolyl.

The term “associating with” refers to a condition of proximity between achemical entity or compound, or portions thereof, and a binding pocketor binding site on a protein. The association may be non-covalent(wherein the juxtaposition is energetically favored by hydrogen bondingor van der Waals or electrostatic interactions) or it may be covalent.

The language “biological activities” of a compound of the inventionincludes all activities elicited by compound of the invention in aresponsive cell. It includes genomic and non-genomic activities elicitedby these compounds.

“Biological composition” or “biological sample” refers to a compositioncontaining or derived from cells or biopolymers. Cell-containingcompositions include, for example, mammalian blood, red cellconcentrates, platelet concentrates, leukocyte concentrates, blood cellproteins, blood plasma, platelet-rich plasma, a plasma concentrate, aprecipitate from any fractionation of the plasma, a supernatant from anyfractionation of the plasma, blood plasma protein fractions, purified orpartially purified blood proteins or other components, serum, semen,mammalian colostrum, milk, saliva, placental extracts, acryoprecipitate, a cryosupernatant, a cell lysate, mammalian cellculture or culture medium, products of fermentation, ascites fluid,proteins induced in blood cells, and products produced in cell cultureby normal or transformed cells (e.g., via recombinant DNA or monoclonalantibody technology). Biological compositions can be cell-free. In apreferred embodiment, a suitable biological composition or biologicalsample is a red blood cell suspension. In some embodiments, the bloodcell suspension includes mammalian blood cells. Preferably, the bloodcells are obtained from a human, a non-human primate, a dog, a cat, ahorse, a cow, a goat, a sheep or a pig. In preferred embodiments, theblood cell suspension includes red blood cells and/or platelets and/orleukocytes and/or bone marrow cells.

The term “chiral” refers to molecules which have the property ofnon-superimposability of the mirror image partner, while the term“achiral” refers to molecules which are superimposable on their mirrorimage partner.

The term “diastereomers” refers to stereoisomers with two or morecenters of dissymmetry and whose molecules are not mirror images of oneanother.

By “agent” is meant a polypeptide, polynucleotide, cell, or fragment, oranalog thereof, small molecule, or other biologically active molecule.

The term “enantiomers” refers to two stereoisomers of a compound whichare non-superimposable mirror images of one another. An equimolarmixture of two enantiomers is called a “racemic mixture” or a“racemate.”

The language “improved properties” refers to any activity associatedwith a responsive MRI contrast agent of the invention that reduces itstoxicity and/or enhances its effectiveness in sensing or detectingbiologically relevant metal ions in vitro or in vivo. In one embodiment,this term refers to any qualitative or quantitative improved property ofa compound of the invention, such as reduced toxicity.

The term “optionally substituted” is intended to encompass groups thatare unsubstituted or are substituted by other than hydrogen at one ormore available positions, typically 1, 2, 3, 4 or 5 positions, by one ormore suitable groups (which may be the same or different). Such optionalsubstituents include, for example, hydroxy, halogen, cyano, nitro,C₁-C₈alkyl, C₂-C₈ alkenyl, C₂-C₈alkynyl, C₁-C₈alkoxy, C₂-C₈alkyl ether,C₃-C₈alkanone, C₁-C₈alkylthio, amino, mono- or di-(C₁-C₈alkyl)amino,haloC₁-C₈alkyl, haloC₁-C₈alkoxy, C₁-C₈alkanoyl, C₂-C₈alkanoyloxy,C₁-C₈alkoxycarbonyl, —COOH, —CONH₂, mono- ordi-(C₁-C₈alkyl)aminocarbonyl, —SO₂NH₂, and/or mono ordi(C₁-C₈alkyl)sulfonamido, as well as carbocyclic and heterocyclicgroups. Optional substitution is also indicated by the phrase“substituted with from 0 to X substituents,” where X is the maximumnumber of possible substituents. Certain optionally substituted groupsare substituted with from 0 to 2, 3 or 4 independently selectedsubstituents (i.e., are unsubstituted or substituted with up to therecited maximum number of substituents).

The term “isomers” or “stereoisomers” refers to compounds which haveidentical chemical constitution, but differ with regard to thearrangement of the atoms or groups in space.

The term “obtaining” as in “obtaining a compound” is intended to includepurchasing, synthesizing or otherwise acquiring the compound.

Responsive MRI Contrast Agents

In certain embodiments, the invention features ¹⁹F-based responsive MRIcontrast agent(s) for detecting and/or sensing Ca²⁺ ions. The Ca²⁺responsive MRI contrast agents include, for example, a compound ofFormula (I), or a salt or ester thereof:

Wherein

R¹, each independently, is C₁₋₃ alkyl,

X is —COOH or

andR², each independently, is C₁₋₃ alkyl.

In one embodiment, each of R¹s in Formula (I) is —CH₂—. In anotherembodiment, X in Formula (I) is

Exemplified Ca²⁺ responsive MRI contrast agents include, such as,

or a salt or ester thereof.

As a specific example, the Ca²⁺ responsive MRI contrast agent usedherein is2,2′,2″,2′″-(2,2′-(ethane-1,2-diylbis(oxy))bis(4-fluoro-2,1-phenylene))bis(azanetriyl)tetraaceticacid (alias, 1,2-bis-[2-bis(carboxymethyl)amino-5-fluorophenoxy]ethane;“5F-BAPTA”), or a salt or ester thereof.

Another example of the Ca²⁺ responsive MRI contrast agent is

or a salt or ester thereof.

Also featured herein are ¹⁹F-based Zn²⁺ responsive MRI contrast agents.In certain embodiments, the Zn²⁺ responsive MRI contrast agent is acompound of Formula (II), or a salt or ester thereof:

Wherein

G is C or N;

m is 1, 2, or 3;

X and Y, each independently, are selected from the group of H, —C₁₋₃alkyl-COOH, —C₁₋₆alkyl, —C₀₋₃alkyl-aryl, and —C₀₋₃alkyl-heteroaryl,wherein said aryl moiety and said heteroaryl moiety are substituted byF, and are further optionally substituted by one or more alkoxy and/orC₁₋₃alkyl.

One embodiment of Formula (II) provides that G is N.

In a certain embodiment, at least one of X and Y in Formula (II) is afluorine-substituted —C₀₋₃alkyl-heteroaryl. The heteroaryl can be, forexample, pyridyl and thienyl.

In still another embodiment, at least one of X and Y is —C₁₋₃alkyl-COOH.

In particular embodiments, the Zn²⁺ responsive MRI contrast agentsinclude compounds of the following formulae, or a salt or ester thereof:

Wherein

m, n, and k, each independently, are 1, 2, or 3; and R is —C₁₋₃ alkyl.

The invention also features other Zn²⁺ responsive MRI contrast agents,such as, a compound of Formula (X), or a salt or ester thereof:

Wherein R₁ and R₂, independently, are alkoxy or C₁₋₃alkyl.

Also provided herein are ¹⁹F-based responsive MRI contrast agent(s) fordetecting and/or sensing Mg²⁺ ions. The Mg²⁺ responsive MRI contrastagents of the invention include, for example, a compound of Formula(XI), or a salt or ester thereof:

Wherein R₃ is H, —C₁₋₃ alkyl, or

and R₄ is H, or —C₁₋₃ alkyl.

The invention also provides ¹⁹F-based responsive MRI contrast agent(s)for detecting and/or sensing Fe²⁺ ions. The Fe²⁺ responsive MRI contrastagents of the invention include, for example, a compound of Formula(XII) to (XIV), or a salt or ester thereof:

In certain embodiments, the invention features the use of a salt orester of a compound of the above formulae. Suitable salts that can beused include those well known in the art (see, e.g., Berge et al. (1977)“Pharmaceutical Salts”, J. Pharm. Sci. 66:1-19). Such a salt can be aninorganic salt (e.g., a sodium salt, a potassium salt, and a cesiumsalt, and etc.) and an organic salt. The esters used herein arepharmaceutically acceptable esters. In particular embodiments, theinvention features acetoxymethyl (AM) esters acetate esters of thecompounds of the above formulae.

It is desired that the ¹⁹F-based responsive MRI contrast agents areprepared and administered in good cell permeable forms.

As an example, 5F-BAPTA can be used and/or administered in one of thefollowing salt/ester forms:

all of which are cell permeant.

The invention also provides ¹⁹F-based responsive MRI contrast agent(s)for detecting and/or sensing multiple metal ions simultaneously (e.g.,K⁺, Na⁺, Ca²⁺, Mg²⁺, Cu²⁺, Fe²⁺ and Zn²⁺). The multiple metal ionresponsive MRI contrast agents of the invention include, for example, acompound of Formula (XV), or a salt or ester thereof:

wherein X is selected from the group consisting of F and one or more ofF, Cl, Br, and I, and n is 2-4. In another embodiment, X is F, and n is1-4. In a preferred embodiment, X is F and n is 2.

Also included herein are stereoisomers (including regio-isomers,diastereomers, and enantiomers), hydrates, and solvates of the compoundsprovided supra.

Methods and Kits Metals are essential for sustaining all forms of life,but alterations in their cellular

homeostasis are connected to severe human disorders, including cancer,diabetes and neurodegenerative diseases (Nat Chem Biol, 2008; 4:168-175). For example, during transplanting human islet in a diabetestherapy, the vast majority of the islet are beta cells secreting insulinand the main ions that could be detected to determine theirfunctionality are Calcium and zinc (FIG. 3). FIG. 3 demonstrates:

a) Glucose is transported into the beta cell by type 2 glucosetransporters (GLUT2). Once inside, the first step in glucose metabolismis the phosphorylation of glucose to produce glucose-6-phosphate. Thisstep is catalyzed by glucokinase—it is the rate-limiting step inglycolysis, and it effectively traps glucose inside the cell;

b) As glucose metabolism proceeds, ATP is produced in the mitochondria.

c) The increase in the ATP:ADP ratio closes ATP-gated potassium channelsin the beta cell membrane. Positively charged potassium ions (K⁺) arenow prevented from leaving the beta cell.

d) The rise in positive charge inside the beta cell causesdepolarization.

e) Voltage-gated calcium channels open, allowing calcium ions (Ca²⁺) toflood into the cell.

f) The increase in intracellular calcium concentration triggers thesecretion of insulin via exocytosis.

Many metal ion indicators are well characterized as well as commerciallyavailable, especially for optical-imaging based approaches.

The invention relates to ¹⁹F-CEST-MRI methods for detecting and/sensingmetal ions in a biological sample or tissue. Specifically, the inventionprovides a non-invasive means of detecting and/or sensing free metalions in a deep tissue.

The CEST contrast mechanism has also been applied to generate MRI-basedcontrast from reporter genes. The process in FIG. 2 demonstrates how theCEST contrast is generated, which is generally through the dynamicexchange process between an exchangeable proton of the biomarker ofinterest and the surrounding water protons. To detect such agents, themagnetization of some of their exchangeable protons is nullified byapplying a selective radiofrequency saturation pulse at the specificresonance frequency (chemical shift) of the target protons. Due toexchange of the “saturated” agent protons with surrounding waterprotons, the net water signal is reduced thus enhancing the MRIcontrast.

The inventors have discovered that chemical saturation transfer isapplicable for ¹⁹F-based MRI. For example, the inventors found that Ca²⁺could be monitored with specificity and high sensitivity with iCEST MRIusing 5F-BAPTA, e.g., Ca²⁺ could be detected in the presence ofcompetitive metal ions. It is believed that the responsive agent (¹⁹F)is the contrast generator (¹⁹F).

In a particular embodiment, the invention presents a novel approach forspecifically sensing the presence of Ca²⁺ in which the amplificationstrategy of chemical exchange saturation transfer (CEST) is combinedwith the broad range in chemical shifts found in ¹⁹F NMR to obtain MRimages of Ca²⁺. The chemical shift change (Δω) of ¹⁹F upon binding ofCa²⁺ to a difluoro derivative of[1,2,-bis(oaminophenoxy)ethane-N,N,—N′,N′,tetra-acetic acid], also namedas 5F-BAPTA, by RF labeling at the bound-19F frequency,ω_([Ca-5F-BAPTA]), and detecting the label transfer to the free-¹⁹Ffrequency, ω_(5F-BAPTA). Through the substrate binding kinetics, thesignal of Ca²⁺ onto free 5F-BAPTA is amplified. Thus, the method of theinvention enables an indirect detection of low Ca²⁺ concentrations withhigh sensitivity.

In one aspect, provided herein is a method of obtaining a magneticresonance (MR) image of a metal ion in a biological sample or tissue,said method comprising

a) introducing a ¹⁹F-based responsive magnetic resonance imaging (MRI)contrast agent to the biological sample or tissue containing the metalion; and

b) imaging the biological sample or tissue using a chemical exchangesaturation transfer (CEST)-based MRI technique.

Another aspect of the invention provides a method of detecting orsensing a metal ion in a biological sample or tissue comprisingbackground ions. The method comprises

a) introducing to the biological sample or tissue ¹⁹F-based responsivemagnetic resonance imaging (MRI) contrast agents, wherein at least oneof the ¹⁹F-based responsive MRI contrast agents is bound to the metalion to produce a chelation complex;

b) radiofrequency (RF) labeling of ¹⁹F frequency in said chelationcomplex; and

c) detecting label transfer to ¹⁹F frequency in a ¹⁹F-based responsiveMRI contrast agent free of metal ion chelation.

The methods of the invention can be applied to many metal ions. Inparticular embodiments, the metal ion is a divalent metal ion (e.g.,Ca²⁺, Zn²⁺, Mg²⁺, Fe²⁺, Cu²⁺, Ba²⁺, Sr²⁺, Cd²⁺, Pb²⁺, Co²⁺ and Ni²⁺).One embodiment provides that the metal ion is Ca²⁺, Zn²⁺, Mg²⁺, or Fe²⁺,or a combination thereof.

The methods of the invention can also be applied to other metal ions,such as, Fe³⁺, Mn³⁺, K⁺, Na⁺, Cu²⁺, Cu⁺, Gd³⁺, and Eu³⁺, or acombination thereof.

According to the invention, the useful ¹⁹F-based responsive magneticresonance imaging (MRI) contrast agents include those provided supra.Alternatively, other ¹⁹F-based fluorescent metal ion sensors known inthe art can also be used in the invention.

As above discussed, the invention presents a novel method of detectingor sensing Ca²⁺ in a biological sample or tissue comprising backgroundions, said method comprising

a) introducing to said biological sample or tissue a ¹⁹F-derivative of1,2,-bis(o-aminophenoxy)ethane-N,N,—N′,N′,tetra-acetic acid (BAPTA), ora salt or ester thereof to obtain a chelation complex containing themetal ion and the ¹⁹F-derivative; and

b) detecting a chemical shift change of ¹⁹F through a ¹⁹F NMR.

In one particular embodiment, the invention relates to the use of5F-BAPTA as a ¹⁹F-based responsive MRI contrast agent for detectingand/or sensing metal ions (especially, Ca²⁺).

The invention thus offers a novel method for monitoring Ca²⁺ by usingMRI, specifically, the method combines the conventional amplificationstrategy and exchange sensitivity of CEST with the Δω specificity of the¹⁹F frequency in free and bound substrate to obtain MR images of calciumbinding kinetics.

It is discovered that BAPTA and its derivatives share high (>10⁵)selectivity for Ca²⁺ over Mg²⁺ of EGTA but are very much less affectedby pH changes and are faster at taking up and releasing Ca²⁺(Biochemistry 19(1980); 2396). ¹⁹F-NMR magnetization transfer between5F-BAPTA and its complexes was presented in literature (such as, NMR InBiomedicine, 7 (1994), 330-338).

Compared to ¹H-based approach, the methods of the invention offeradvantages, such as, large range of ¹⁹F chemical shifts (about 20 timesthat of ¹H); and high sensitivity of ¹⁹F chemical shifts to the detailsof the local environment

The invention also provides kits for imaging free metal ions in abiological sample or tissue. The kit of the invention includes one ormore ¹⁹F-based responsive MRI contrast agents (e.g., 5F-BAPTA) of theinvention, and instructions for imaging free metal ions (e.g., Ca²⁺) ina biological sample or tissue.

The invention also provides a method with improved specificity andsensitivity for imaging a metal ion in a biological sample or tissuecontaining background ions, comprising the steps of chelating said metalion with a ¹⁹F-based responsive magnetic resonance imaging (MRI)contrast agent to obtain a chelation metal complex, and imaging saidchelation metal complex using a CEST-based MRI technique.

The iCEST approach of the invention can be further extended to designingof novel responsive agents for molecular and cellular MRI applications.Any potential novel responsive agents are assessed by an optical assay(using multi-well plates) for their potentiality for the iCEST approach.

The invention also includes a method of chemically modifyingcommercially available chelates for metal ion detection, comprisingsteps of designing and synthesizing novel chelates, and screening thenovel metal ions for their usefulness in the iCEST approach.

Certain design criteria for creating metal responsive MRI contrastagents can be found in Que et al. (Chem Soc. Rev. 2010, 39, 51-60) andHyman et al. (Coordination Chemistry Reviews, 256 (2012), 2333-2356).

Further, the ¹⁹F-based iCEST MRI approach of the invention can also bewidely applied in other types of applications, for example, as a methodfor imaging and/or analysis reactive oxygen species and otherbiologically relevant compounds, or as a diagnostic method for variousdiseases or conditions.

Example General Methods Material:

5F-BAPTA (5,5′-difluoro BAPTA): The difluoro derivative of the tetrapotassium salt of [1,2,-bis(o-aminophenoxy)ethane-N,N,—N′,N′,tetra-acetic acid], 5F-BAPTA, was purchased from Biotium, Inc. (Hayward,Calif., USA).

Sample Preparation:

5F-BAPTA was dissolved in Hepes buffer (40 mM) to a final concentrationof 10 mM and the pH was adjusted to the following values: 5.6, 6.0, 6.4,6.8, 7.0, 7.2, and 7.6. Stock 10 mM solutions of CaCl₂, MgCl₂, and ZnCl₂were prepared in 40 mM Hepes buffer. Five μL of the stock solution wasadded to 1 mL of 10 mM 5F-BAPTA resulting in a 200:1 ratio (10 mM: 50μM) between the free 5F-BAPTA and the M²⁺ ion following by a pHadjustment. One mL of each sample (with adjusted pH) was transferredinto a 8 mm NMR tube within a 25 mm NMR tube in order to center thesample in the coil for the MRI experiments.

¹⁹F NMR Experiments:

¹⁹F-NMR spectra were acquired with 11.7 T NMR scanner (Bruker) with adedicated ¹⁹F coil (470 MHz). Samples contained 5F-BAPTA (5.0 mM) M²⁺(Ca2+, Mg2+ or Zn2+, 0.5 mM), 5-Fluoro-Cytosine (5-FC, 0.5 mM) and D2O(10%) that was used for signal lock. 5FC was assigned as an internalreference with a fixed frequency of −47.0 ppm. The data show that thefrequency of free 5FBAPTA is affected by pH, while that of bound5F-BAPTA is not.

MRI Experiments:

MRI experiments were performed on a vertical 16.4 T scanner (BrukerAvance system) at 37° C. A 25 mm birdcage radiofrequency coil was usedto acquire both ¹H and ¹⁹F MR images by sweeping the coil frequency fromproton (700 MHz) to fluorine (658.8 MHz) frequency.

¹H-MRI:

A RARE sequence was used to acquire the ¹H-MR images with the followingparameters: TR/TE=5,000/7.7 ms, RARE factor=8, 1 mm slice thickness,FOV=4×4 cm, matrix size=128×128, resolution=0.3125×0.3125 mm, and 1average (NA=1).

Magnetization Transfer Ratio (MTR) Images, iCEST Images:

The saturation transfer effect on the free 5F-BAPTA, i.e., the MTR oriCEST, was calculated for each voxel in the image by using a Lorentzianline shape fitting as described in Jones et al. (Magn Reson Med 2012,67, 1579), and Liu et al. (Molecular imaging 2012, 11, 47).

Relaxation Times:

The same image geometry as in the ¹⁹F-CEST experiments was used for thedetermination of T1 and T2 of the imaged samples. For T1 measurements, asaturation recovery experiment was performed with TR values of 61, 214,395, 615, 899, 1296 1964, and 4961 ms. A spin-echo experiment (TR=5000ms) with multiple echoes was performed with variable echo times (TE=S,10 15, 20, 25, 30, 35, and 40 ms) to determine the T2 value of eachsample.

Bloch Equation Simulations:

Numerical solutions to the six Bloch equations including directsaturation of free 5F-BAPTA were obtained as described previously(McMahon, M. T. et al., Magnetic Resonance in Medicine 2006, 55, 836).The relaxation parameters for ¹⁹F used in the Bloch equations wereR₁=1/T₁ with bound calcium (R_(1b))=0.71 s⁻¹, R₂ bound calcium(R_(2b))=29 s⁻¹, whereas the R₁ and R₂ values of free 5F-BAPTA (R_(1f)and R_(2f)) were determined experimentally through inversion-recoveryand saturation-recovery experiments as a function of pH on solutionscontaining 20 mM 5F-BAPTA as listed in Table 1 as follows:

TABLE 1 pH Δω (ppm) T₂ (ms) T₁ (ms) 5.6 2.1   6^(a) 715 6.0 4.0 10 7156.4 5.0 11 715 6.8 5.6 25 715 7.0 5.9 32 715 7.2 6.2 38 715 7.6 6.2 N.D715 ^(a)Estimated from the Bloch equation fitting N.D. = Not Determined

¹⁹F-iCEST Experiments:

A modified RARE sequence (TR/TE=4,000/3.4 ms, RARE factor=4, 10 mm slicethickness, FOV=4×4 cm, matrix size=32×32, resolution=1.25×1.25 mm, andNA=8) including a magnetization transfer (MT) module (B1=3.6 μT) wasused to acquire CEST-weighted images from −7.2 to +7.2 ppm around theresonance of the ¹⁹F atoms at the free 5F-BAPTA, which was assigned as 0ppm. Saturation time (tsat) was either 1500 ms to 2000 ms as indicatedin the text.

¹⁹F-iCEST of 500 nM of Ca²⁺:

An aqueous solution containing 0.5 mM of 5F-BAPTA and 500 nM in Hepesbuffer (40 mM, pH=7.2) was transferred into a 20 mm NMR tube, which waslocated within a 25 mm NMR tube in order to centralize the sample in thecoil for the MRI experiment. The same parameters used for ¹⁹F-iCESTexperiments were used except the followings: TR=3000 ms, RARE=8, FOV=6.0cm (resolution=1.875×1.875 mm), and 168 averages.

¹⁹F-CEST Experiments with 5F-BAPTA

¹⁹F-CEST experiments were performed on solutions containing1,2-Bis-[2-bis(carboxymethyl)amino-5-fluorophenoxy]ethane (5F-BAPTA),with and without divalent cations (M²⁺).

Experiments were performed on an 11.7 T NMR spectrometer (Bruker).5F-BAPTA (AnaSpec, Inc.) was dissolved in 40 mM HEPES buffer (pH=7.0) toa concentration of 5 mM, and CaCl₂ was dissolved to a final Ca²⁺concentration of 50 μM.

¹⁹F-CEST spectra (z-spectra) were acquired with a saturation transfersequence consisting of a saturation pulse (B1=4.7 μT, 4 sec) withvariable offset (from −7.5 to +7.5 ppm relative to the 5FBAPTA frequencyset at 0 ppm). Experiments were performed without (room temperature, RT)and with sample heating (37° C.). The MTRasym=100×(S_(−Δω)−S_(+Δω))/S₀was computed at different offsets Δω, where S₀ is the 5F-BAPTA signalwithout saturation.

MRI:

Imaging experiments were performed on a 16.4 T MRI scanner (Bruker).5F-BAPTA was dissolved to 10 mM in 40 mM HEPES, pH=7.0, with or without100 μM of M²⁺ (Mg²⁺, Zn²⁺, or Ca²⁺).

¹⁹F-CEST images were acquired using a continuous wave presaturationpulse (B₁=3.6 μT, 3 sec) followed by a multi-echo MRI pulse sequence(RARE, rare factor 4, TR/TE=6000/10 ms). FOV of 4×4 cm, matrix 32×32 andslice thickness 10 mm Mean ¹⁹F-CEST spectra were derived after B₀correction for each voxel using MatLab. MTRasym plots and maps werecalculated.

FIG. 16a illustrates the chemical shift offsets (Δω) of the 5FBAPTA atthe ¹⁹F NMR frequencies upon complexation with various divalent cations(M²⁺) (Smith et al., Ptoc Natl. Acad Sci, USA 80, 1983, 7178). FIG. 16bdepicts the dynamic exchange process between the free 5FBAPTA andCa²⁺-bound 5F-BAPTA, i.e., [Ca-5FBAPTA], allowing indirect detection oflow Ca2+ concentrations by using saturation transfer (FIG. 16c ).

FIG. 16c shows the ¹⁹F-CEST and MTRasym plots of 5 mM 5F-BAPTA in thepresence of 50 μM Ca²⁺ in HEPES buffer (pH=7.0). The dynamic ionexchange process between 5F-BAPTA and [Ca-5FBAPTA], which increases withtemperature, results in an observed increase in the MTRasym value atΔω=5.8 ppm (Δω of [Ca-5F-BAPTA]) resulting in a change in the 5F-BAPTAsignal.

Evaluation of the Sensitivity of iCEST Contrast Approach

The iCEST contrast of 5FBAPTA solutions (pH=7.2) at different ratios ofCa²⁺ to 5F-BAPTA were examined (χCa, FIGS. 7a and 12). As clearly shownin FIG. 12, Ca²⁺ is easily detected with iCEST MRI at χCa=1:1000, where11% contrast is observed in the Zspectrum for this phantom. The sameamplification using iCEST contrast was obtained when 0.5 mM 5F-BAPTA wasused to detect 500 nM Ca²⁺ (FIG. 7b ), showing the potential of iCEST tosense low Ca²⁺ concentrations.

This study shows for the first time that spatial information of Ca²⁺ andMg⁺² levels can be obtained using amplification of the sensitivity byiCEST with 5F-BAPTA as the ion indicator. One advantage of using5F-BAPTA as an MRI responsive agent for detecting metal ions over¹H-MRI²⁶ or ¹²⁹Xe-MRI²⁷ based probes is that no attachment of a contrastenhancer is required. The ¹⁹F atoms serve on the chelates as theresponsive group as well as contrast generators.

The study shows the potential of exploiting the iCEST imaging conceptusing ¹⁹F-MRI, as 1:2000 concentration ratios are amplified to 1:20changes in ¹⁹F signal (FIGS. 7a and 12), i.e., an amplification factorof ˜100 for a kex of 190 s⁻¹. Moreover, the signal from lowconcentration solutes [Ca²⁺-5F-BAPTA] is amplified through saturationtransfer onto the signal of the high concentration free 5F-BAPTA. Sincethis contrast is dependent on χCa, lower concentrations of Ca²⁺ can bedetected through simply reducing the free 5F-BAPTA concentrations. Thisis an advantage of the iCEST approach, since this feature is notavailable for ¹H CEST imaging, which is based on water.

Finally, the unique Δω found for each [M²⁺-5FBAPTA] and the diversity ofthe obtained kex may be exploited for multi-ion MR imaging approaches inwhich each ion generates iCEST contrast with an identifiable amplitudeand Δω.

¹⁹F-CEST Properties of 5F-BAPTA in the Presence of Different Metal Ions

FIG. 4a illustrates the dynamic exchange process between free 5F-BAPTAand its complex with M²⁺, [M²⁺-5F-BAPTA]. Upon M²⁺ binding, there is a¹⁹F chemical shift change (Δω) for 5F-BAPTA. If the exchange rate (kex)between M²⁺-bound and free 5F-BAPTA is fast on the NMR time scale(Δω<<k_(ex)), no peak can be resolved. When the k_(ex) is sufficientlyslow at the field strength used, a well-defined peak is observed for the[M²⁺-5F-BAPTA] resonance as is shown for Zn²⁺ (Δω>>kex) and Ca²⁺(Δω>kex).

The observed Δω's are typical and unique for each ion that is complexedby 5F-BAPTA and ranges from a few ppm in the cases of Ca²⁺, Zn²⁺, Ba²⁺,Sr²⁺, Cd²⁺, Pb²⁺ and others to tens of ppm upon binding of Fe²⁺, Co²⁺and Ni²⁺ (Smith et al., Proc Natl Acad Sci USA 1983, 80, 7178;Kirschenlohr, H. L. et al., Biochem J 2000, 346 Pt 2, 385). Thedissociation constant (K_(d)) of [M²⁺-5F-BAPTA] is different for eachM²⁺, and as a result so is the k_(ex) for the process in FIG. 4a . TheZn²⁺-5F-BAPTA resonance (FIG. 4 b, 4.1 ppm) is sharper than theCa²⁺-5F-BAPTA resonance (FIG. 4 b, 6.2 ppm), which is correlated withtheir reported differences in K_(d).

The ¹⁹F-CEST properties of 5F-BAPTA in the presence of Ca²⁺(slow-to-intermediate k_(ex)), Zn²⁺ (very slow k_(ex)) and Mg²⁺ (fastkex) were determined on a 16.4 T. MRI scanner and are summarized in FIG.5 for two different pH values, i.e. 7.2 (FIG. 5 a-c) and 6.4 (FIG. 5d-f). At these concentrations, a pronounced saturation transfer contrastwas detected in the Ca²⁺ containing solutions (FIGS. 5a,d ) but not inthe Zn²⁺ or Mg²⁺ containing solutions (FIG. 5b,e or FIG. 5c,f ,respectively).

Importantly, a broad asymmetry is observed at very high fractional Mg²⁺concentrations (FIG. 10b , χ(5FBAPTA/Mg)=50:1), which peaks at ˜1.8 ppm,a frequency much lower than Ca2+ (FIG. 10a ).

Interestingly, the Δω between [Ca-5F-BAPTA] and free 5F-BAPTA was foundto be dependent on pH (FIGS. 5, 6, 8, 9 and 11 and Table 1), but thek_(ex) between [Ca-5F-BAPTA] and 5F-BAPTA was preserved for all examinedpH values as determined by Bloch simulations (190±10 s⁻¹, FIGS. 5 and 8)(McMahon, M. T. et al., Magn Reson Med 2006, 55, 836) These results arein a good agreement with a previous report showing that the binding ofCa²⁺ was unaffected at pH 6-8 using ¹⁹F-MRS.

¹⁹F-NMR spectra collected with an internal reference revealed that uponpH change, the frequency of the free 5F-BAPTA shifts but not thefrequency of bound M²⁺-5F-BAPTA (FIG. 9). The T2 values of 5F-BAPTA arealso sensitive to pH as can be seen by the broadening in the Z-spectra(FIGS. 5 and 8; and Table 1). The T2-value changes seem to be dependenton 5F-BAPTA protonation and not exchange rate dependent based on theobservation that the same Z-spectra line widths were found for solutionscontaining Mg²⁺ (Δω<<kex) and Zn²⁺ (Δω>>kex). FIG. 6 shows MR images ofthe samples that have been used in this study, i.e., 10 mM of 5F-BAPTAand 50 μM of M²⁺.

As expected no difference in MR contrast was observed between thesamples when using conventional ¹H-MRI or ¹⁹F-MRI. However, contrary tothe Mg²⁺- or Zn²⁺-containing samples, which did not generate iCESTcontrast at this concentration, a large iCEST contrast was detected forthe Ca²⁺ containing sample when a saturation pulse (B₁=3.6 μT/2000 ms)was applied at the appropriate frequency offset of the [Ca²⁺-5F-BAPTA]complex, i.e., Δω=6.2 ppm (pH=7.2) and Δω=5.0 ppm (pH=6.4). FIG. 11shows the dependence of Δω on pH, with Δω ranging from 2.1 ppm to 6.2ppm for pH values of 5.6 to 7.2.

In addition, iCEST images were acquired for solutions containingmixtures of Ca²⁺ and Mg²⁺ (50 μM Ca²⁺, 200 μM Mg²⁺) and Ca²⁺ and Zn²⁺(50 μM Ca²⁺, 50 μM Zn²⁺) with 10 mM BAPTA at pH 7.2. The iCEST contrastproduced by the Ca²⁺ was still significant (˜22%) at Δω=6 ppm for allmixtures (FIG. 10). Although high Mg²⁺ generates iCEST contrast atΔω=1.8 ppm (FIG. 10 10 a-b) the larger Δω, smaller k_(ex) of[Ca-5F-BAPTA] and its much higher iCEST contrast makes this approachbetter for Ca²⁺ sensing (FIG. 10b , amplification factor=×10 for Mg²⁺,×100 for Ca²⁺).

Ca²⁺ Specificity of 5F-BAPTA

The iCest approach regarding Ca²⁺ specificity was studied by using5F-BAPTA as a metal ion indicator. The results were presented in FIGS.13 and 14. As shown in FIGS. 13 a-c, 5F-BAPTA demonstrates a high Ca²⁺specificity in a Zn²⁺ background. FIGS. 14 a-c, 5F-BAPTA alsodemonstrates a high Ca²⁺ specificity in a Mg²⁺ background.

The ¹⁹F-CEST MR images shown in FIG. 17 clearly demonstrate thespecificity of the iCEST approach. On conventional ¹⁹F-MRI (FIG. 17a ),no difference in contrast could be observed for the different tubes (noM²⁺, Mg²⁺, Zn²⁺, and Ca²⁺), as this detects only the free 5F-BAPTA. On¹⁹F-CEST MRI, only the Ca²⁺ containing solution generated contrast (FIG.17b ). Here, the saturation pulse was applied at Δω=5.8 ppm from the5FBAPTA resonance (0 ppm). FIG. 17c shows the mean ¹⁹F-CEST MTRasym plotfor the tube containing 5FBAPTA in the presence of Ca²⁺.

The variance in the Δω of [M²⁺-5F-BAPTA] complex for each examined M²⁺makes ¹⁹F-CEST MRI more specific than relaxation based ¹H MRImethodologies. The specificity is comparable to that of PARACEST agentsused for Ca⁺² and Zn⁺² binding (G. Angelovski et al., Bioorg Med Chem19, 1097 (2011); R. Trokowski et al., Angew Chem Int Ed Engl 44, 6920(2005)), but no paramagnetic agent is needed. The higher saturationtransfer effect for 5F-BAPTA obtained at 37° C. as compared to RT (FIG.16c ) is due to the faster ion exchange rate between 5F-BAPTA and[Ca-5F-BAPTA]. This confirms that the observed effect is due to thedynamic binding kinetics process.

It is contemplated that the specificity of the ¹⁹F-CEST method proposedhere is not just due to the different Δω values of the examined ions(FIG. 16a ), but also due to the different dissociation constants(K_(d)) between 5FBAPTA and the divalent metal complex [M²⁺-5F-BAPTA],which determines the exchange rate (k_(ex)) and therefore the ¹⁹F-CESTcontrast. The K_(d) for Ca²⁺ results in slow to intermediate k_(ex) onthe NMR time scale (H. Gilboa et al., NMR Biomed 7, 330 (1994)), whilethe K_(d) values for Mg²⁺ and Zn²⁺ result in k_(ex) values that are toofast and too slow, respectively, preventing the observation of signalchanges in the ¹⁹F-CEST experiment.

Simultaneous Detection of Multiple Metal Ions Using a Single 19F-iCESTProbe

In another embodiment of the invention, the ¹⁹F iCEST MRI approach maybe extended by chemical modification of a BAPTA derivative,5,5′,6,6′-tetrafluoro-BAPTA (TF-BAPTA, AG Scientific, Inc.), that altersthe binding kinetics of metal ions and their chelates, enabling specificand simultaneous detection of Zn²⁺ and Fe²⁺. TF-BAPTA has the followingstructure:

Without being bound by theory, it is believed that the above-shownacetate form allows the TF-BAPTA compound to enter into desired targetcells, where the acetate may be hydrolyzed and the free acid form isreleased. The free acid form may allow binding of the ions to bedetected. Accordingly, one of skill in the art will appreciate that theacetate form may typically be used to facilitate the transport ofTF-BAPTA into the cell for in vivo imaging application, whereas ex vivoimaging applications will typically involve an appropriate free acidform of the molecule.

Methods

Experiments were performed on solutions containing TF-BAPTA and multiplebiologically relevant metal ions (K⁺, Na⁺, Ca²⁺, Mg²⁺, Cu¹²⁺, Fe²⁺ andZn²⁺) at 37° C. and pH=7.4. MR Spectroscopy as follows: TF-BAPTA wasdissolved to a final concentration of 5 mM, and ions were add at 500 μM.Using 5-Fluorocytosine (5-FC) as an internal ¹⁹F reference, ¹⁹F-NMRspectra were acquired (11.7 T NMR spectrometer, Bruker). MRI:Experiments were performed on a 17.6 T MRI scanner (Bruker). TF-BAPTAwas dissolved to 10 mM with 200″ AM of ion. A RARE (factor 8) sequencewas used to acquire 1H MRI (TR/TE=5,000/7.7 ms, 1 mm slice, FOV=2×2 cm,matrix size=128×128). For ¹⁹F MRI, the center frequency (O1) was set atthe frequency of the 19F atom at the 6 position (0.0 ppm) of TF-BAPTA(FIG. 1a ), while signal from the ¹⁹F located at the 5 position ofTF-BAPTA (FIG. 1a -b, 4.5 ppm downfield) was suppressed. A modified RAREsequence (TR/TE=4,000/3.4 ms, RARE factor=16, 6 mm slice, FOV=2×2 cm,matrix size=32×32, and a saturation pulse B1=1.2, 2.4 or 3.6 ItT/2 s)were used to acquire ¹⁹F iCEST. Mean ¹⁹F Z-spectra (iCEST spectra), wereobtained after B₀ correction. CEST contrasts, i.e., the magnetizationtransfer ratio (MTR) images were calculated after Lorentzian line shapefitting.

FIG. 18a illustrates the chemical structure of TF-BAPTA. FIG. 18b showsthe ¹⁹F NMR spectra of TF-BAPTA in the presence of Zn²⁺ or Fe²⁺, alongwith the peak assignments. As shown previously for Ca²⁺ binding toTF-BAPTA (London et al., 1994, Am J Physiol 266:1313), the downfield NMRpeaks (10.5 ppm for Zn²⁺-TF-BAPTA and 39.5 ppm for Fe²⁺-TF-BAPTA) arethe observed Δωs of the ¹⁹F atom at the 5 position (purple, FIG. 18a ),while the upfield Δωs are related to the ¹⁹F atom at the 6 position(green, FIG. 18a ). Note that all other examined ions did not revealadditional peaks in the ¹⁹F NMR spectrum of TF-BAPTA, except for Ca²⁺which exchanges extremely fast (30,000 s-1) with TF-BAPTA for Δω=9.7ppm, shifting the peak at the 5 position upon its addition (London etal., 1994, Am J Physiol 266:1313). FIG. 18c shows the ¹H-MRI and ¹⁹F-MRIof 7 tubes containing 10 mM TF-BAPTA and 200 μM of added ion, withoutany changes in ¹H or ¹⁹F MR contrast. However, ¹⁹F iCEST showed a cleardifferential MR contrast between the Zn²⁺ and Fe²⁺ containing samples.The iCEST: −2.8 ppm and iCEST: −18 ppm images represent the ¹⁹F iCESTcontrast obtained when the saturation pulse (B1=3.6 μT/2 s) was appliedat Δω=−2.8 ppm and Aw=−18 ppm, respectively. These Δω values werecorrelated with the Δωs in the ¹⁹F NMR spectra upon addition of Zn²⁺ orFe²⁺, respectively (see FIG. 18b ). FIG. 19a-b shows the corresponding¹⁹F-iCEST-spectra from the samples containing either Zn²⁺ (FIG. 19a ) orFe²⁺ (FIG. 19b ). The dynamic ion exchange between TF-BAPTA and[M²⁺-TF-EBAPTA] results in an observed iCEST effect for both ions atΔω=−2.8 ppm for [Zn²⁺-TF-BAPTA] and at Δω=−18 ppm for [Fe²⁺-TF-BAPTA].Using Bloch simulations (solid lines in FIG. 19a-b , using a two poolmodel) the exchange rate (k_(ex)) between free and bound TF-BAPTA isestimated to be ˜20 s-1 for both ions. Interestingly, when both ionswere combined with TF-BAPTA (FIG. 19c ), two distinctive peaks wereobtained in the iCEST spectra, which was supported by the Blochsimulations (using a three-pool model). These data confirm that Zn²⁺ andFe²⁺ can be monitored simultaneously using a single iCEST probe.

The high sensitivity of the ¹⁹F NMR spectrum Δω values for changes inchemical environment together with the specificity of these Δωs forcertain metal ions allows the development of novel responsive contrastagents for ¹⁹F-iCEST. By adding ¹⁹F atoms to the 6 position of 5F-BAPTA,which previously allowed only detection of Ca²⁺ using iCEST (Bar-Shir etal., 2013, J Am Chem Soc 135:12164; Gilboa et al., 1994, NMR Biomed7:330), to obtain TF-BAPTA (FIG. 18a ), it became possible to detectboth Zn²⁺ and Fe²⁺. Adding one ¹⁹F atom to the BAPTA backbonedramatically changes the binding properties of TF-BAPTA (London et al.,1994, Am J Physiol 266:1313). At the same time, the added ¹⁹F-atominduces k_(ex) values that allow detection of Zn²⁺ and Fe²⁺ with¹⁹F-iCEST MRI. Although other ¹H MRI probes for the detection of Zn²⁺can be used (Lubag et al., 2011, PNAS), the specificity of iCEST tosimultaneously detect different ions using the same sensor represents anew concept for the rational design of novel MRI probes. While theobserved k_(ex) between bound and free TF-BAPTA is only 20 s-1 for bothions, and higher CEST effect could be obtained for higher k_(ex), (vanZijl et al., 2011, Magn Reson Med 65:927) it was still possible todetect a 200 μM concentration with a 10 mM signal strength. Withoutbeing bound by theory, it is believed that this may be due to the natureof iCEST that allows the reduction of the concentration of the ¹⁹F-iCESTprobe to a detectable molar ratio, a feature that is not available for¹H-CEST, which is based on water. However, further chemicalmodifications of the fluorinated probe may result in higher k^(ex) andtherefore in higher iCEST contrast.

Although a preferred embodiment of the invention has been describedusing specific terms, such description is for illustrative purposesonly, and it is to be understood that changes and variations may be madewithout departing from the spirit or scope of the following claims.

INCORPORATION BY REFERENCE

All patents, published patent applications and other referencesdisclosed herein are hereby expressly incorporated by reference in theirentireties by reference.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents of the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

What is claimed is:
 1. A method of obtaining a magnetic resonance (MR)image of a metal ion in a biological sample or tissue, comprising a)introducing a ¹⁹F-based responsive magnetic resonance imaging (MRI)contrast agent to said biological sample or tissue; and b) imaging saidbiological sample or tissue using a chemical exchange saturationtransfer (CEST)-based MRI technique.
 2. A method selected from the groupconsisting of a method of detecting or sensing a metal ion in abiological sample or tissue comprising background ions, comprising a)introducing to said biological sample or tissue ¹⁹F-based responsivemagnetic resonance imaging (MRI) contrast agents, wherein at least oneof the ¹⁹F-based responsive MRI contrast agents is bound to the metalion to produce a chelation complex; b) radiofrequency (RF) labeling of¹⁹F frequency in said chelation complex; and c) detecting label transferto ¹⁹F frequency in a ¹⁹F-based responsive MRI contrast agent free ofmetal ion chelation; a method of detecting or sensing Ca²⁺ in abiological sample or tissue comprising background ions, said methodcomprising a) introducing to said biological sample or tissue a¹⁹F-derivative of 1,2,-bis(o-aminophenoxy)ethane-N,N,—N′,N′,tetra-aceticacid (BAPTA), or a salt or ester thereof to obtain a chelation complexcontaining the metal ion and the ¹⁹F-derivative; and b) detecting achemical shift change of ¹⁹F through a ¹⁹F NMR; a method of imaging ametal ion in a biological sample or tissue comprising background ions,comprising the steps of chelating the metal ion with a ¹⁹F-basedresponsive magnetic resonance imaging (MRI) contrast agent to obtain achelation metal complex, and imaging said chelation metal complex usinga CEST-based MRI technique; and a method of obtaining a magneticresonance (MR) image of two or more metal ions in a biological sample ortissue, comprising a) introducing a ¹⁹F-based responsive magneticresonance imaging (MRI) contrast agent to said biological sample ortissue; and b) imaging said biological sample or tissue using a chemicalexchange saturation transfer (CEST)-based MRI technique.
 3. The methodof claim 2, wherein said method comprises detecting a chemical shiftchange of ¹⁹F through a ¹⁹F NMR.
 4. The method of claim 1, wherein saidmetal ion is a divalent metal ion.
 5. The method of claim 1, whereinsaid metal ion is selected from the group of Ca²⁺, Zn²⁺, Mg²⁺, Fe³⁺,Fe²⁺, Mn³⁺, K⁺, Na⁺, Cu²⁺, Gd³⁺, and Eu³⁺, or a combination thereof. 6.The method of claim 5, wherein said metal ion is Ca²⁺.
 7. The method ofclaim 6, wherein said ¹⁹F-based responsive MRI contrast agent(s) is acompound of Formula (I), or a salt or ester thereof:

Wherein R¹, each independently, is C₁₋₃ alkyl, X is —COOH or

and R², each independently, is C₁₋₃ alkyl.
 8. The method of claim 7,wherein each of R¹s is —CH₂—, X is


9. The method of claim 7, wherein said ¹⁹F-based responsive MRI contrastagent is selected from the group consisting of

or a salt or ester thereof and

or a salt or ester thereof.
 10. The method of claim 9, wherein said¹⁹F-based responsive MRI contrast agent is2,2′,2′,2′″-(2,2′-(ethane-1,2-diylbis(oxy))bis(4-fluoro-2,1-phenylene))bis(azanetriyl)tetraaceticacid (5F-BAPTA) or a salt thereof.
 11. (canceled)
 12. The method ofclaim 5, wherein said metal ion is Zn²⁺.
 13. The method of claim 12,wherein said ¹⁹F-based responsive MRI contrast agent is a compound ofFormula (II), or a salt or ester thereof:

Wherein G is C or N; m is 1, 2, or 3; X and Y, each independently, areselected from the group of H, —C₁₋₃ alkyl-COOH, —C₁₋₆alkyl,—C₀₋₃alkyl-aryl, and —C₀₋₃alkyl-heteroaryl, wherein said aryl moiety andsaid heteroaryl moiety are substituted by F, and are further optionallysubstituted by one or more alkoxy and/or C₁₋₃alkyl.
 14. The method ofclaim 13, wherein G is N.
 15. The method of claim 14, wherein at leastone of X and Y is a fluorine-substituted —C₀₋₃alkyl-heteroaryl.
 16. Themethod of claim 15, wherein said ¹⁹F-based responsive MRI contrast agentis a compound of one of Formula (III) to (VI):

Wherein m, n, and k, each independently, are 1, 2, or 3; and R is —C₁₋₃alkyl; or a salt or ester thereof.
 17. The method of claim 14, whereinat least one of X and Y is —C₁₋₃ alkyl-COOH.
 18. The method of claim 17,wherein said ¹⁹F-based responsive MRI contrast agent is a compound ofone of Formula (VII) to (IX), or a salt or ester thereof:

Wherein R is —C₁₋₃ alkyl.
 19. The method of claim 12, wherein said¹⁹F-based responsive MRI contrast agent is a compound of Formula (X), ora salt or ester thereof:

Wherein R₁ and R₂, independently, are alkoxy or C₁₋₃alkyl.
 20. Themethod of claim 5, wherein said metal ion is Mg²⁺.
 21. The method ofclaim 20, wherein said ¹⁹F-based responsive MRI contrast agent is acompound of Formula (XI), or a salt or ester thereof:

Wherein R₃ is H, —C₁₋₃ alkyl, or

and R₄ is H, or —C₁₋₃ alkyl.
 22. The method of claim 5, wherein saidmetal ion is Fe²⁺.
 23. The method of claim 22 wherein said ¹⁹F-basedresponsive MRI contrast agent is a compound of Formula (XII) to (XIV),or a salt or ester thereof:


24. (canceled)
 25. The method of claim 2, wherein said ¹⁹F-derivative ofBAPTA is 5F-BAPTA.
 26. A kit comprising one or more ¹⁹F-based responsiveMRI contrast agents of Formulae (I) to (XIV), and instructions forimaging free metal ions in a biological sample or tissue.
 27. The kit ofclaim 26, wherein said ¹⁹F-based responsive MRI contrast agent is5F-BAPTA, and said free metal ions comprise Ca²⁺. 28-29. (canceled) 30.The method of claim 2, wherein the two or more metal ions are selectedfrom the group consisting of K⁺, Na⁺, Ca²⁺, Mg²⁺, Cu²⁺, Fe²⁺ and Zn²⁺.31. The method of claim 2, wherein the ¹⁹F-based responsive magneticresonance imaging (MRI) contrast agent is5,5′,6,6′-tetrafluoro-1,2,-bis(o-aminophenoxy)ethane-N,N,—N′,N′,tetra-acetate.32. The method of claim 2, wherein the 19F-based responsive magneticresonance imaging (MRI) contrast agent has the structure of Formula XV:

wherein X is F and one of F, Cl, Br, or I, and n is 2-4.
 33. The methodof claim 32, wherein X is F and n is 2.